One-step purification of functional human and rat pancreatic alpha cells

Martin Köhler, Elisabetta Daré, Muhammed Yusuf Ali, Subu Surendran Rajasekaran, Tilo Moede, Barbara Leibiger, Ingo B. Leibiger, Annika Tibell, Lisa Juntti-Berggren, Per Olof Berggren

Research output: Contribution to journalArticle

18 Citations (Scopus)

Abstract

Pancreatic alpha cells contribute to glucose homeostasis by the regulated secretion of glucagon, which increases glycogenolysis and hepatic gluconeogenesis in response to hypoglycemia. Alterations of glucagon secretion are observed in diabetic patients and exacerbate the disease. The restricted availability of purified primary alpha cells has limited our understanding of their function in health and disease. This study was designed to establish convenient protocols for the purification of viable alpha cells from rat and human pancreatic islets by FACS, using intrinsic cellular properties. Islets were isolated from the pancreata of Wistar rats or deceased human organ donors. Dispersed islet cells were separated by FACS based on light scatter and autofluorescence. Purity of sorted cells was evaluated by immunocytochemistry using hormone specific antibodies. Relative hormone expression was further determined by quantitative RT-PCR. Viability was determined by Annexin V and propidium iodide staining and function was assessed by monitoring cytoplasmic free Ca 2+ concentration ([Ca 2+] i) using Fura-2/AM. We developed species-specific FACS gating strategies that resulted in populations consisting mainly of alpha cells (96.6 ± 1.4%, n = 3 for rat; 95.4 ± 1.7%, n = 4 for human, mean ± SEM). These cell fractions showed ∼5-fold and ∼4-fold enrichment (rat and human, respectively) of glucagon mRNA expression compared to total ungated islet cells. Most of the sorted cells were viable and functional, as they responded with an increase in [Ca 2+] i upon stimulation with l-arginine (10 mM). The majority of the sorted human alpha cells responded also to stimulation with kainate (100 μM), whereas this response was infrequent in rat alpha cells. Using the same sample preparation, but a different gating strategy, we were also able to sort rat and human populations enriched in beta cells. In conclusion, we have simplified and optimized a method for the purification of rat alpha cells, as well as established a novel approach to separate human alpha cells using neither antibodies nor dyes possibly interfering with cellular functions.

Original languageEnglish
Pages (from-to)209-219
Number of pages11
JournalIntegrative Biology
Volume4
Issue number2
DOIs
StatePublished - Feb 1 2012
Externally publishedYes

Fingerprint

Glucagon-Secreting Cells
Purification
Rats
Glucagon
Islets of Langerhans
Hormones
Propidium
Antibodies
Fura-2
Kainic Acid
Annexin A5
Arginine
Glycogenolysis
Coloring Agents
Gluconeogenesis
Health
Availability
Glucose
Hypoglycemia
Messenger RNA

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry

Cite this

Köhler, M., Daré, E., Ali, M. Y., Rajasekaran, S. S., Moede, T., Leibiger, B., ... Berggren, P. O. (2012). One-step purification of functional human and rat pancreatic alpha cells. Integrative Biology, 4(2), 209-219. https://doi.org/10.1039/c2ib00125j

One-step purification of functional human and rat pancreatic alpha cells. / Köhler, Martin; Daré, Elisabetta; Ali, Muhammed Yusuf; Rajasekaran, Subu Surendran; Moede, Tilo; Leibiger, Barbara; Leibiger, Ingo B.; Tibell, Annika; Juntti-Berggren, Lisa; Berggren, Per Olof.

In: Integrative Biology, Vol. 4, No. 2, 01.02.2012, p. 209-219.

Research output: Contribution to journalArticle

Köhler, M, Daré, E, Ali, MY, Rajasekaran, SS, Moede, T, Leibiger, B, Leibiger, IB, Tibell, A, Juntti-Berggren, L & Berggren, PO 2012, 'One-step purification of functional human and rat pancreatic alpha cells', Integrative Biology, vol. 4, no. 2, pp. 209-219. https://doi.org/10.1039/c2ib00125j
Köhler M, Daré E, Ali MY, Rajasekaran SS, Moede T, Leibiger B et al. One-step purification of functional human and rat pancreatic alpha cells. Integrative Biology. 2012 Feb 1;4(2):209-219. https://doi.org/10.1039/c2ib00125j
Köhler, Martin ; Daré, Elisabetta ; Ali, Muhammed Yusuf ; Rajasekaran, Subu Surendran ; Moede, Tilo ; Leibiger, Barbara ; Leibiger, Ingo B. ; Tibell, Annika ; Juntti-Berggren, Lisa ; Berggren, Per Olof. / One-step purification of functional human and rat pancreatic alpha cells. In: Integrative Biology. 2012 ; Vol. 4, No. 2. pp. 209-219.
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